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AutomaticAd1918 OP t1_iy3cc1a wrote

Reply to comment by HotDadBod1255 in How exactly does CRISPR-CAS9 insert new genes? by AutomaticAd1918

Ooh I see based on the answers, I've also understood that instead of "inserting" genes, in general we really just break off a part of DNA, then allow the cell to repair it and just hope it repairs it in a way that we would like or we can give a template so the cell can more likely repair it in a desired way?

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HotDadBod1255 t1_iy3g7wt wrote

Correct! In ex vivo applications we really need an insertion mechanism.

For in vivo, there are a lot of diseases we can treat by just knocking out the gene and not inserting anything.

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AutomaticAd1918 OP t1_iy3gt52 wrote

Ooohh I see how are genes knocked out or shut off? Do we introduce another protein to stop the cell from repairing it?

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HotDadBod1255 t1_iy3lmrj wrote

Nope, it's just the Cas9 protein and the guide RNA. The guide tells Cas9 precisely where to make cuts in the DNA. The part that's cut is naturally removed and digested by the cell. Then when that happens, the cell's natural repair mechanism takes the two open ends of DNA and connects them together.

As you might guess there's some work required to figure out dosing. To low of a dose and you probably won't edit enough cells, so the un-cut gene will persist. Too high of a dose and you're probably going to harm the patient or cell.

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